We hypothesize that pathological vascular calcification is caused by the local activation of pericytes to express their osteoblast "program" and produce calcified matrix in the context of the atherosclerotic lesion. Calcified plaque is an important risk factor for thromboembolism, myocardial infarction, and stroke - major healthcare issues. Pericytes are mesenchymal cells that reside in arteries and form gap junctions with endothelial cells in the microvasculature. Pericytes have multipotent differentiation potential, forming osteoblasts, vascular smooth muscle cells, adipocytes, chondrocytes, and fibroblasts depending on paracrine regulatory signals from adjacent cells, cytokines, growth factors, other soluble factors, and extracellular matrix. We will use primary rat and human pericytes to investigate the regulation of calcification in vitro. Because there are no pericyte cell lines, we will also immortalize human pericytes with forced expression of human telomerase reverse transcriptase (hTERT-pericyte) in an effort to develop a reliable, clinically relevant research model. Aim 1 characterizes the role of soluble factors and culture conditions that control differentiation of pericytes into the calcifying osteoblast-like phenotype, and attempts to more clearly define this phenotype by gene array methods. Aim 2 defines the ligand-dependent signaling pathways that are critical for pericyte differentiation and calcification. Here we will test our second hypothesis that pericytes are the target for cholesterol-lowering statin drugs, through modulation of cholesterol-rich preassembled membrane signaling complexes (caveolae and lipid rafts) and/or reduced prenylation of accessory signaling proteins. Cholesterol-lowering statin drugs have strong efficacy in treating atherosclerosis, as well as important actions on bone mass. Aim 3 studies the cell-cell interactions between pericytes and some of their principal neighbors in atherosclerotic lesions (endothelial cells, macrophages, and foam cells) that may initiate and sustain pericyte osteogenic differentiation. The in vitro work should yield a body of detailed information on factors and gene expression changes that regulate pericyte-mediated calcification. In Aim 4 these markers will then be investigated in vivo in calcifying atherosclerotic lesions of hypercholesterolemic WHHL rabbits undergoing statin drug therapy, and in human pathological specimens. We believe that this Project will provide a foundation for new therapeutic strategies to treat cardiovascular calcification and atherosclerosis. This Project responds to RFA-HL-01-014 by linking investigators in 3 areas critical to the advancement of knowledge of vascular calcification: vascular cell biology (pericytes), bone cell biology (osteoblast differentiation, membrane signaling, mineralized matrix, RANKL actions on macrophage lineage), and atherosclerosis and statin research (rabbit and human studies). We believe this proposal addresses vascular calcification with powerful technology, original insights, and novel avenues of investigation.